Friday, 29 April 2011

I see London, I see France, I see…Drosophila’s internal structure

This isn't going to be a long, lengthy post, but rather a short note on an interesting paper just published in Frontiers in Systems Neuroscience, detailing a neat advancement in ultramicroscopy.

As you probably already know, Drosophila is perhaps the most commonly used model organism in genetics. When a geneticist used Drosophila for a genetic screen, he generates mutants and then scans the progeny for particular desired phenotypes. For instance, if you're interested in a gene involved in the development of limbs, you'd generate mutants and then look for ones that have mutated legs. External phenotypes like this are pretty easy to observe, even without a microscope, but it's a bit harder when it comes to internal structures. What if your gene of interest is involved in forming the gut, or a particular set of muscles? One way you could go about observing internal structures would be to dissect your flies, but this has its limitations – it requires good manual dexterity, and has the added risk of tearing, ripping or otherwise mutilating your specimen. You could use in situ staining or florescence microscopy, but what you end up with is a flat 2-dimensional image that might not reveal all the details that would be present in three dimensions. Using a confocal microscope will give you good resolution, but generally use high magnifications that will not allow you to view your whole specimen at once. The paper by Jährling et al details a technique using ultramicroscopy that allows for an entire 3D reconstruction of a specimen, complete will internal structures visualized in situ.

The basic procedure goes like this: they began by "chemically clearing" their specimens – that is, using a series of chemical washes and incubations, they removed almost all colour from their specimens. They were left with flies which were nearly transparent. This would allow the internal structures to be visualized. The specimens were then mounted on an ultramicroscope, and using a laser, they took a series of 597 images, beginning at the top and moving down through the vertical plane. Once the images had been taken, they used specialized software to layer the images on top of one another to reconstruct a 3-dimensonal model. Since the flies were transparent, the model allowed for the visualization of internal structures as well as the specimen's surface. Using this technique, you can easily visualize internal structures that might be of interest to you without ever having to dissect your specimen or rely on 2-dimensional imaging techniques.

This technique really becomes powerful when coupled with fluorescent microscopy. Imagine you're convinced that your gene of interest plays a role in the development of the fly's gut. Attach GFP to a gut-specific promoter, insert the construct into your flies and then image them. What you'd get is a perfect 3D model of the fly's gut, easily distinguishable from surrounding tissue. Any phenotypic effects would be easy to observe! Using this technique, you could easily, quickly (the authors state that it takes about 30 minutes from start to finish) and reliably visualize any internal structure you wish. Pretty cool, no?
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References:
Jährling N, Becker K, Schönbauer C, Schnorrer F and Dodt H-U (2010). Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy. Front. Syst. Neurosci. 4:1. doi: 10.3389/neuro.06.001.2010